PRODUCTION OF OMEGA- 3 FATTY ACID CONCENTRATES FROM FISH AND ALGAL OILS AND THEIR STRUCTURAL ASSESSMENT BY NMR

Transcription

1 PRDUCTIN F MEGA- 3 FATTY ACID CNCENTRATES FRM FISH AND ALGAL ILS AND THEIR STRUCTURAL ASSESSMENT BY NMR Kralovec, JA 1, Weijie W 1, Barrow CJ 2 and Reyes-Suarez E 1 1 cean Nutrition Canada Ltd. Dartmouth, Nova Scotia, B2Y 4T6, Canada; 2 School of Life and Environmental Science, Faculty of Science and Technology, 1 Pigdons Road, Geelong, Victoria 2127, Australia 1

2 LC-PUFA, MEGA-3 vs. MEGA-6 H 3 C α-linolenic acid (ALA, C18:3, n-3) H 3 C linoleic acid (C18:2, n-6) CH CH H 3 C arachindonic acid (ARA, C18:4, n-6) CH H 3 C eicosapentaenoic acid (, C20:5, n-3) H 3 C docosahexaenoicoic acid (, C22:6, n-3) CH CH LC-PUFAs are categorized by the position of the 1 st double bond counting from the CH 3 -terminal Location of the 1 st double bond dictates biological activity All double bonds are CH 2 -interruped mega-3 series starts with α-linolenic acid (ALA,18:3n-3) mega-6 series starts with linoleic acid (LA,18:2n-6) The most studied omega-3 FA are and Calder. Schweiz. Zeit. Ernährungmedizin 2009, 5,

3 PRDUCTIN F / CNCENTRATES STARTING MEGA-3 IL SELECTIN The richest and the cheapest source of / is fish oil Starting oils with right FA profiles and / content ~30%! anchovy/sardine (18/12TG, for high level concentrates) tuna (05/25TG, for high level concentrates) CRE MEGA-3 CNCENTRATIN TECHNLGIES Fish oils are in the form of triglycerides Winterization Cold Solvent Precipitation Urea Clathration CR 1 CR 3 CR 2 Ethanolysis + Fractional Distillation Ethyl Ester (EE) Concentrates Enzyme-assisted Concentration Triglyceride (TG) Concentrates 3

4 EE CNCENTRATES ETHANLYSIS AND DISTILLATIN Ethanolysis Distillation TG EE EE 30% 30% 60% Ethanolysis - TG EE Distillation EE Splitting TG molecules lowers the boiling points! -TG bp ~ 910 o C, vs. -EE bp ~ 450 o C) Popular technology towering over the other options Medium chain or medium chain EE residues or -EE residues or -EE Glycerol backbone or glycerol = CI 1.67, output CI, capital investment Economic advantage of large throughput! 4

5 CNCENTRATIN STRATEGY A ETHANLYSIS / DISTILLATIN / GLYCERLYSIS GLYCERLYSIS Ethanolysis - Distillation TG EE EE TG EE TG FFA H RC RCR 1 + H i - R 1 H RC H RC R, a mixture of C14-C24 hydrocarbon residues,, primarily those of and/or R1, H or Et; i, CALB, C, vacuum CALB Immobilized Candida antarctica lipase B (CALB) in Packed Bed Reactor The reaction is driven forward by EtH or water removal under vacuum 5

6 PACKED BED REACTR Trap (condenser) Evaporation tank Heated stirred tank Vacuum pump Distributor Reactor Pump Pump 6

7 MANUFACTURING F TG CNCENTRATES Green technologies (free or immobilized lipases) Packed bed reactors Reaction mixture cycling trough PBR First to introduce food grade immobilized lipase for EE TG conversion! EE i FFA ii TG i: CALB-L, EtH/H2, 50 oc, vacuum ii: CALB-FPX66, glycerol, 70 oc, vacuum ~ 80 reactions on 1 batch of immobilized. enzyme! 7

8 CNCENTRATIN STRATEGY B HYDRLYSIS AND () INSERTIN Hydrolysis / Insertion - TG DG TG Hydrolysis: Lipo-JD, P buffer ph7.5, 40 o C, 42 h; / insertion: CALB, / source, 70 o C, 24h TIME CURSE F 18/12TG HYDRLYSIS in glyceride in glyceride in FFA in FFA / Content (% Candida rugosa lipase Total FFA in reaction mixture (%) 8

9 PSITINAL DISTRIBUTIN (PD) F / IN TG ILS It is very likely that the oxidative stability and thus also sensory qualities of / rich TG oils and their TG concentrates correlate with the positional distribution of fatty acid residues, primarily and in TG molecules. FACTS AND STRATEGY Complexity Fish oil contains 30 to 60 different fatty acids Assuming 30 and taking into account positional distribution, theoretically different TGs CR 1 CR 2 Chromatography Complex mixtures, need for non-aqueous conditions Mass spectrometry CR 3 If R 1 R 2, C 2 is chiral Neutral compounds - poor ionization, difficulty adding reagents to mobile phase for adduct formation NMR Complex mixtures, limited data in the literature 9

10 SYNTHESIS F STANDARDS SYMMETRIC H (CH 2 ) 14 CH 3 EDCl, DMAP (CH 2 ) 14 CH 3 CH 2 Cl 2 ASYMMETRIC Method A H 3 C(H 2 C) 14 (CH 2 ) 14 CH 3 1,3-dipalmitate H 1,2-dipalmitate (CH 2 ) 14 CH 3 EDCl, DMAP CH 2 Cl 2 H 3 C(H 2 C) 14 (CH 2 ) 14 CH 3 (CH 2 ) 14 CH 3 Palmitic acid EDCl, DMAP CH 2 Cl 2 Method B EDCl, DMAP Amberlyst H H CH 2 Cl 2 H MeH, CH 2 Cl 2 Wijesundera et. al. J. Amer. il Chem. Soc. 2007, 84,

11 PSITINAL DISTRIBUTIN F AND BY NMR 13 C NMR (preferred) Better resolution (larger spectral range) than 1 H NMR, although lower sensitivity CARBNYL (C-1) CARBNS (preferred) Carbonyls from sn-1,3- and sn-2 chains form clusters of peaks Within each cluster, chemical shifts differences between acyl chains occur as a result of the proximity to a neighboring double bond Unable to discriminate between sn-1 and sn-3 positions 11

12 PSITINAL DISTRIBUTIN F AND BY NMR Carbonyl region of the 125 MHz 13 C NMR spectrum of a mixture of TG standards of seven fatty acids BASIC FACTS Carbonyls from sn-1,3 chains resonate at higher chemical shifts than sn-2 chains Within each cluster carbonyls from saturated chains resonate at a higher chemical shift sn-1,3 sn ppm Chemical shift (ppm) Carbonyl chemical shifts of acyl chains that differ in double bond group C-1 C-1 γ to an olefinic carbon Sat Δ9 Δ8 Δ7 Δ6 Δ5 Δ4 Position of the first double bond relative to the carbonyl carbon Chemical shifts are in a direct correlation with the distance of the carbonyl carbon to the first double bond Unsaturation on the γ-position ( 4, ) induces a greater shift on carbonyl chemical shifts; carbonyls resonate in a unique position, which allows a straightforward assignment 12

13 PSITINAL DISTRIBUTIN F AND BY NMR INVESTIGATED FISH ILS 18/12TG (sardine oil) Re-esterified 18/12TG 05/25TG (tuna oil) QUANTITATIVE 13 C NMR Under full-decoupling conditions ne build up may differ for carbonyls of sn-1,3 and sn-2 chains Sn-1,3/sn-2 area ratios were measured for and carbonyls as a function of the relaxation delay for 18/12TG (100mg/0.7mL CDCl 3 ) Areas ratio Variation of the carbonyl areas ratio: A/A for and A/A(sn- 1,3) for as a function of the relxation delay in a full-decoupled sequence (C=) (C=) Area ratios for both and carbonyls remain constant after 12s delay (working range) Relaxation delay (s) 13

14 PSITINAL DISTRIBUTIN F AND BY NMR Carbonyl region of the 125 MHz 13 C NMR spectrum of 18/12TG oil (100 mg/ 0.7 ml CDCl 3 ) Carbonyl region of the 125 MHz 13 C NMR spectrum of Re-esterified 18/12TG oil (100 mg/ 0.7 ml CDCl 3 ) Sat Monounsat Sat Sat Monounsat Sat STA Monounsat STA STA Monounsat STA ppm ppm Sat Carbonyl region of the 125 MHz 13 C NMR spectrum of 0525TG tuna oil (100 mg/ 0.7 ml CDCl 3 ) Monounsat Sat Monounsat ppm 14

15 PSITINAL DISTRIBUTIN F AND BY NMR Molar percentages of individual chains in sn-1,3 an sn-2 glycerol positions (fish oil TG 18/12) RE-ESTERIFICATIN F 18/12TG % Sat Mono STA Ethanolysis - (No concentration of or ) % /Total % /Total The distribution of saturated and monounsaturated chains as well stearidonic acid (STA) remains unaffected % Molar percentages of individual chains in sn-1,3 and sn-2 glycerol positions (TG re-esterified) Sat Mono STA The distribution of and chains is affected that was mostly in sn-1,3 is randomized on re-esterification that was mostly in sn-2 is 5 0 % /Total % /Total located mostly in sn-1,3 on reesterification 15

16 PSITINAL DISTRIBUTIN F AND BY NMR 05/25TG TUNA IL % 40 Molar percentages of individual chains in sn-1,3 and sn-2 glycerol positions (tuna oil) Sat Mono Saturated and monounsaturated chains are preferably located in sn-1,3 positions is present in equal amounts 10 5 in both sn-1,3 and sn-2 positions 0 % /Total % /Total shows a slight preference for the sn-2 position 16

17 SUMMARY The evidence for the beneficial effects of omega-3 fatty acids FA, particularly and is undisputable. Science and innovation are the major drivers of dietary supplement and pharmaconutrient industry. To deliver high quality omega-3 products the replacement of traditional chemistry by enzymatic technologies for manufacturing omega-3 based products is becoming more and more important. The understanding of the positional distribution (PD) of the FA residues in the oils is of crucial importance since PD might be important for the stability, sensory and biological effects. NC is leading the way in implementation of sophisticated manufacturing technologies and thorough understanding of physiochemical properties of omega-3 fatty acids. 17

18 THANK YU! 18